DNA Replication (With Diagram) | Molecular Biology
Let us make an in-depth study of the DNA replication:- Learn about: 1. Basic Features of DNA Replication 2. Mechanism of DNA Replication 3. Meselson and Stahl Experiment 4. Enzymes of DNA Replication 5. Formation of Replication Forks & Replication Bubbles and Others.
Genetic material is always nucleic acid and it is always DNA except some viruses. DNA is the storehouse of genetic information. This information is in the form of nucleotide sequence called genetic code. This information is copied and transcribed into RNA molecules. This information (genetic code) is for specific sequence of amino acids. The RNA then synthesizes proteins, which are specific sequence of amino acids, by a process called translation. In 1956 Francis Crick called this pathway of flow of genetic information as the Central Dogma.
Both transcription and translation are unidirectional. Proteins never serve as template for RNA synthesis. But sometimes RNA acts as a template for DNA synthesis (reverse transcription), Example is RNA viruses (HIV virus).
Genetic information present in double stranded DNA molecule is transmitted from one cell to another cell at the time of mitosis and from parent to progency by faithful replication of parental DNA molecules. DNA molecule is coiled and twisted and has enormous size. This imposes several restrictions on DNA replication. DNA molecule must be uncoiled and the two strands must be separated for the replication process.
Basic Features of DNA Replication:
All genetically relevant information of any DNA molecule is present in its sequence of bases on two strands. Therefore the main role of replication is to duplicate the base sequence of parent DNA molecule. The two strands have complementary base pairing. Adenine of one strand pairs with thymine of the opposite strand and guanine pairs with cytosine. This specific complementary base pairing provides the mechartism for the replication.
The two strands uncoil and permanently separate from each other. Each strand functions as a template for the new complementary daughter strand. The base sequence of parent or old strand directs the base sequence of new or daughter strand. If there is adenine in the parent or old strand, complementary thymine will be added to the new strand. Similarly, if there is cytosine in the parent strand, complementary guanine will be copied into the new daughter strand. Maintenance of integrity of genetic information is the main feature of replication.
Mechanism of DNA Replication:
Mechanism of DNA replication is the direct result of DNA double helical structure proposed by Watson and Crick. It is a complex multistep process involving many enzymes.
It involves the origin of replication. Before the DNA synthesis begins, both the parental strands must unwind and separate permanently into single stranded state. The synthesis of new daughter strands is initiated at the replication fork. In fact, there are many start sites.
The next step involves the addition of new complementary strands. The choice of nucleotides to be added in the new strand is dictated by the sequence of bases on the template strand. New nucleotides are added one by one to the end of growing strand by an enzyme called DNA polymerase. There are four nucleotides, deoxyribrnucleotide triphosphates dGTP, dCTP, dATP, dTTP present in the cytoplasm.
All the end termination reactions occur. Duplicated DNA molecules are separated from one another.
The purpose of DNA replication is to create two daughter DNA molecules which are identical to the parent molecule.
DNA Replication is Semi-Conservative:
Watson and Crick model suggested that DNA replication is semi-conservative. It implies that half of the DNA is conserved. Only one new strand is synthesized, the other strand is the original DNA strand (template) that is retained. Each parental DNA strand serves as a template for one new complementary strand.
The new strand is hydrogen bonded to its parental template strand and forms double helix. Each of these strands of the double helix contains one original parental strand and one newly formed strand.
Meselson and Stahl Experiment:
Mathew Meselson and Franklin Stahl proved experimentally that parental strands of a helix are distributed equally between the two daughter molecules. They made use of the heavy isotope 15 N as a tag to differentially label the parental strands. E. coli was grown in a medium containing 15 N labeled NH4Cl.
In this way both strands of DNA molecules were labeled with radioactive heavy isotope 15 N in their purines and pyrimidines. Therefore both strands were heavy or HH DNA. The bacteria were then transferred into a medium containing the common non-radioactive nitrogen 14 N, which is a light medium. It was found that after one cell division daughter molecules had one 15 N strand the other 14 N strand. So this is a hybrid molecule, a heavy light of HL.
After the second cell division, out of four molecules, two DNA molecules contained U N(LL). The other two were hybrid molecules (HL). This proves that during replication, one parent strand is conserved and the other new strand is synthesized. Thus DNA replication is a semi-conservative process.
Enzymes of DNA Replication:
The enzymes which take part in replication are able to copy DNA molecules which may contain millions of bases. They perform this function with utmost accuracy and at high speed, even though DNA molecule is highly compact and is bound with proteins. Maintenance of integrity of genetic information is the main feature of replication.
In E. coli, two main enzymes that take part in polymerization are DNA polymerase I and DNA polymerase III. Of these DNA polymerase III is the main enzyme involved in replication. Polymerization involves addition of new nucleotides to a growing strand.
In addition, there is an enzyme DNA polymerase II which takes part in DNA repair. DNA polymerase IV and DNA polymerase V have also been discovered. DNA polymerases are capable of adding 1000 nucleotides per second. The speed of DNA synthesis is known as processivity.
Nucleotide monophosphates, which are building blocks of DNA, cannot be added to the growing strand of DNA. The DNA polymerase can act only on deoxyribose triphosphate nucleotides. They are dATP, d GTP, d CTP and dTTP. Triphosphate nucleotides possess high-energy phosphate bonds.
DNA polymerase I and DNA polymerase III can add new nucleotides at the 3′-OH end of the growing strand. Inorganic pyrophosphate is released. Hydrolysis of pyrophosphate is the driving force for DNA synthesis. Phosphodiester bond is formed between 3′-end of growing strand and 5′-end of first phosphate of incoming nucleotide.
The two DNA strands separate and each acts as a template for the formation of new strand. Polymerization-reaction is dictated by a template strand according to the base pairing rules where if adenine is present in the template, thymine is added to the new strand and guanine pairs with cytosine.
DNA polymerase needs primer to synthesize new strand on it. Primer is a small strand segment which is complementary to the template. It has a free 3′-OH end to which a new nucleotide can be added. In this way part of the new strand is already in place. Primer is hydrogen bonded to the template to form primer: template junction. Primer is short nucleotide strand (oligonucleotide). The primer for both the new strands is RNA primer. The RNA primer is synthesized by an enzyme, primase.
DNA polymerase moves along the template adding nucleotides. Thus DNA is synthesized by extending the 3′-end of the primer. Afterwards, primer is removed by DNA polymerase I and RNAse H.
Leading strand requires only a single RNA primer. On the other hand, discontinuous synthesis of lagging strand requires primer for each Okazaki fragments. For synthesis of lagging strand hundreds of Okazaki fragments with their associated RNA primers are required.
In this way two conditions are necessary for DNA synthesis. They are a template and a primer with 3′-OH end.
DNA Synthesis takes Place in 5′ → 3′ Direction Only:
A new strand of DNA is always synthesized in 5′ → 3′ direction. The free 3′-end enables it to be elongated. Because the two strands are antiparallel, orientation of new growing strand is opposite to the template strand.
DNA Replication is Discontinuous in One Strand:
If the synthesis is to proceed in 5′ → 3′ direction, only one strand can be synthesized in correct 5′ → 3′ direction. As the strands are antiparallel, the other strand will have to be synthesized in 3′ 5′ direction.
This constraint is overcome in an ingenious way. As the two strands unwind and the replication fork grows, one strand is synthesized in 5′ 3′ direction correctly in a continuous manner. This is called leading strand. It grows in the same direction as the replication fork.
Mr. Reiji Okazeki discovered that the other strand is synthesized discontinuously and a little after the leading strand. Therefore this is called lagging strand. The lagging strand also grows in 5′ 3′ direction which is opposite to the direction of replication fork. In lagging strand, the DNA synthesis does not occur continuously but in small fragments, which are called Okazaki fragments. Later these fragments are joined and sealed by the action of DNA ligase enzyme to form a continuous strand.
Okazaki fragments are about 1000-2000 nucleotides long in E. coli and about 100-200 nucleotides long in eukaryotes. Both the strands begin the synthesis starting on a primer segment. Leading strand synthesis starting on a primer proceeds continuously keeping pace with the unwinding of DNA at the replication fork.
Each Okazaki fragment is synthesized on a short RNA primer. DNA polymerase III binds to RNA primer and adds deoxyribonucelotides. Lagging strand proceeds in opposite direction from the fork movement.
The primer ribonucleotides are removed and replaced by deoxyribornucleotides and then joined. The removal of RNA primer is done by exonuclease activity of DNA polymerase I.
Unwinding of Double Helix:
The first step of DNA replication is the unwinding parent double helix molecule so that each strand acts as a template for the new strand. Unwinding mechanism is very complex. Hydrogen bonds between two strands are broken. This is achieved by enzymes called DNA helicases which move along DNA and separate the strands.
DNA helicases bind to the lagging strand template. Strand separation create topological stress like the one produced if the two ends of a coiled rope are pulled apart fc- separation. This forms supercoils in the unreplicated double helix in front of the replication fork. This stress is removed and strands are separated by the action of DNA topoisomerase Topoisomerase I introduces cut or nick in one DNA strand.
Topoisonmerase II remove supercoils by causing double stranded breaks. Some parts of DNA molecule have circle linked as a chain. This structure is called catenane. DNA gyrase is capable of decatenating two circles, thus separating them. In this way these enzymes open up and unwind the DNA helix.
In order to prevent the formation of hydrogen bonds again between two separated strand- the two single strands are coated by Single Strand Binding Proteins (SSB proteins), which stabilize the separated strands.
Leading strand is synthesized continuously but lagging strand is synthesized discontinuously. This is called semi-discontinuous replication.
Replication is Highly Accurate:
Replication takes place with an extraordinary accuracy In spite of all this, errors do occur and wrong nucleotides are added. Approximately one mistake occurs in every 10 1 ” nucleotides added. But mechanisms do exist for the repair of DNA molecule. In fact DNA is the only molecule for which repair mechanisms exist.
Proof Reading or Editing Functions:
DNA polymerase I is a very versatile enzyme. In addition to its ability to polymerize, it also performs repair function. Sometimes a wrong nucleotide is added at the 3′-OH end which fails to base pair with the complementary base on the template strand. Then the polymerization process is stopped. The enzyme moves backward and removes the wrong base by degrading DNA.
Then the polymerization activity resumes and chain growth starts again by adding correct base. It removes only the most recent error. This process is known as 3′ → 5′ exonuclease activity and the enzyme is called proof reading exonuclease. This editing function gives a second chance to DNA polymerase to add correct nucleotide.
Replication in Eukaryotic Cells:
The chromosomal DNA replication occurs only once during S-phase of cell cycle. The basic features of replication in eukaryotic cells are the same as of prokaryotes. Process of polymerization is similar to prokaryotes. New nucleotides are added at 3′-OH end like prokaryotes. But there are some major differences.
The DNA molecule considerably large as compared to bacterial chromosomes. Eukaryotic chromosomes are linear and has free ends. They are organized into complex nucleoproteins chromatin.
Replication in eukaryotes is considerably faster. Replication is completed in three minutes in embryonic cels of drosophilla. Chromosomes of higher organisms have multiple origins of replication and all replication forks proceed bidirectionally.
Eukaryotic DNA has repeated units of replication called replicons. Enormous number of replication units require large number of polymerase enzyme molecules. An animal cell has 20000 – 60000 molecules of polymerase ∝.
Eukaryotes have several types of polymerase enzymes. The main three enzymes are DNA polymerase ∝ DNA polymerase δ and DNA polymerase є. Replication is initiated by DNA polymerase ∝ and DNA polymerase δ and є bring about rapid polymerization because of their high processivity.
Eukaryotic replication also synthesizes end structures or telomeres.
Formation of Replication Forks and Replication Bubbles:
Initiation of replication occurs within the double helix and rarely at the end. Opening up of DNA molecule creates replication bubble. Replication bubble progresses in the form of replication fork in one direction in the case of unidirectional replication and in both directions in bi-directional replications.
In circular DNA of bacteria and linear DNA of eukaryotes, DNA replication proceeds bidirectionarlly starting from a fixed origin of replication. The two replication forks move in opposite directions. Bidirectional replication may have multiple replication forks. They speed up the process of replication.
Eukaryotic chromosomes are very long. In order to speed up the process of replication, a chromosome may have thousands of points of origin of replication (O). They tremendously speed up the process of replication. Bidirectional replication forks proceed in opposite directions and meet the neighbouring replication units, thereby opening the entire chromosome by separating two strands.
Unwinding and Replication of Circular Double Helix DNA of E. Coli:
Most of the bacteria have double stranded circular DNA with no free ends. This poses a problem of unwinding at the time of replication. Replication originates at one point and the two replication forks proceed in opposite directions. The advancing replication forks meet at a point opposite to the point of origin thus opening up the coiled DNA molecule. This is called θ (theta) model of replication.
But the unwinding is a very complex mechanism as the two strands are coiled. The two advancing replication forks make the remaining entire un-replicated portion of DNA overwound. Thus, the un-replicated portion becomes so tightly coiled that the advancing replication forks are not able to advance further.
This is because of positive supercoiling of the un-replicated portion. In E. coli an enzyme called DNA gyrase produces negative super coiling, thus removing the positive supercoiling. This leads to unwinding of the entire double helix circular chromosome of the bacteria.
Newly Synthesized Eukaryotic DNA Immediately Forms Nucleosomes:
Before the replication takes place, the DNA disentangles itself from the nucleosomes. After replication, newly synthesized DNA immediately rejoins the octomers of histone proteins to form nucleosomes.
Large amounts of histones are synthesized during .S’-phase of interphase. Old histones are not lost. Old histones are present on both daughter chromosomes. During replication, nucleosomes are broken down into their components and later reassembled into nucleosomes.
DNA is the storehouse of genetic information. This information in the form of nucleotide sequence called genetic code. This information is transcribed onto RNA which translate this information into sequence of amino acids (Protein). Francis Crick called this central dogma
Genetic information present in double stranded DNA molecule is transmitted from one cell to another cell and to progeny by faithful replication of DNA molecules. The main role of replication is to duplicate the base sequence of parent DNA molecule. The two strands uncoil and permanently separate from each other. Each strand
functions as a template for the new complementary daughter strand.
The base sequence of parent or old strand directs the complementary base sequence of new or daughter strand. Replication includes steps initiation, elongation and termination. DNA replication is semi- conservative. Out of two strands formed, one old or parental strand is retained and the other view strand is synthesized. This was experimentally proved by Meselson and Stahl in E. coli. Main enzyme involved in replication is DNA polymerse III. Other enzymes involved are DNA polymerase I, II, IV and V.
Four kinds of nucleotides are building blocks of DNA. The DNA polymerase acts on dATP, dGTP, dCTP, dTTP. These new nucleotides are added one-by-one at 3′-OH end of the growing strand. DNA polymerase needs a primer to synthesize new strand. Small RNA primer hydrogen bonds with the template. This primer provides free 3′- OH end to add new nucleotides. DNA synthesis takes place in 5′ → 3′ direction.
Therefore only one strand which grows in the direction of replication fork is synthesized in 5′ → 3′ direction. This is called leading strand and is synthesized continuously. The other strand is synthesized in small fragments in 5′ → 3′ direction. These fragments are called Okazaki fragments. Later these fragments are joined and sealed by DNA ligase enzyme. This strand is called lagging strand and is synthesized discontinuously.
Unwinding of DNA Double Helix:
As each strand acts as a template for new strand, the two strands must unwind. Hydrogen bonds between two strands are broken by enzymes called DNA helicases. DNA topoisomearases also help in unwinding. Single strand binding proteins (SSB proteins) bind the unwound single strands to prevent the formation of hydrogen bonds again.
Sometimes wrong bases are added at the 3′-OH end. DNA polymerase I enzyme performs the repair function. It moves backward 3′ → 5′ direction and removes the last base. This is called exonuclease function. Then DNA synthesis resumes is 5′ → 3′ direction.
Replication in Eukaryotic Cells:
The basic features of replication are same in prokaryotes and eukaryotes except some difference. Polymerization is similar in both cases. Eukaryotic chromosomes are much longer and have free ends. Polymerase enzymes are DNA polymeraze α DNA polymerase δ and DNA polymerase є.
Formation of Replication Forks and Replication Bubbles:
Opening up DNA molecule creates replication bubbles which progress in the form of replication fork. Replication fork progresses in one direction in case of unidirectional replication and in both directions in bi-directional replication. Eukaryotic chromosomes are very long, so there may be thousands of points of origin of replication. This speeds up the process of replication tremendously.
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Bioflix Activity Dna Replication Dna Replication Diagram
It shows how both strands of the dna helix are unzipped and copied to produce two identical dna molecules. Each strand is a polymer of dna nucleotides.
Mastering Biology Chapter 16 Rhs Homework
As we all know dna is the genetic code that helps our cells to develop and reproduce in a planned way.
Bioflix activity dna replication dna replication diagram. Before we jump into the process of replication let us take a quick look at the structure of dna. Dna replication 1 of 2. A closer look part a in a dna double helix an adenine of one strand always pairs with an of the complementary strand and a guanine of one strand always pairs with an of the complementary strand.
Pick out one of each of the 4 types of nucleotides 1 a 1 g 1 t and 1 c. Part a nucleotide pairing drag the labels onto the diagram to identify how nucleotides pair up. To download the subtitles srt.
This 3d animation shows you how dna is copied in a cell. Getting back to its structure dna is made up of four nucleotides. Because of which it is called the blueprint of life.
Dna replication nucleotide pairing can you label the way nucleotides pair up in replicating dna. Dna replication dna replication diagram. Activity modified from from dna to protein manipulatives catalog number 6731058 wards scientific.
Start studying dna replication protein synthesis. Google search the pbs title and you can find the website which has links to many informative sites and. Dna polymerase begins synthesizing the lagging stand by adding nucleotides to a short segment of rna 2.
Learn vocabulary terms and more with flashcards games and other study tools. Genetic information present in double stranded dna molecule is transmitted from one cell to another cell and to progeny by faithful replication of dna molecules. Dna replication lagging strand synthesis.
The main role of replication is to duplicate the base sequence of parent dna molecule. Show the dna replication bioflix on mastering biology. After each piece of lagging stand is.
To review dna replication watch this bioflix animation. Dna structure and replication machinery bioflix tutorial part a the chemical structure of dna and its nucleotides the dna double helix is composed of two strands of dna. Labels can be used once more than once or not.
Dna structure replication examining dna structure. It details the latest research as of 2005 concerning the process of dna replication.
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You need to know the basic process of DNA replication and how it relates to the transmission and conservation of genetic information.
DNA (Deoxyrebonucleic acid) is a large molecule that directs how proteins will be assembled. DNA is a nucleic acid made up of nucleotides. Each nucleotide consists of a phosphate group, a 5 carbon sugar deoxyribose, and a nitrogenous base adenine, thymine, guanine and cytosine.
Base rule pair Adenine with Thymine and Guanine with Cytosine
DNA Replication occurs during S phase of interphase before cell division.
DNA replication results in two identical daughter molecules, each consisting of one old (original) strand and one newly-synthesized strand.
First, the two original strands separate. Then, DNA polymerases add complementary nucleotides to each strand. Because of the strictness of base-pairing rules the result is always the formation of two DNA molecules that are identical to the original DNA molecule.
The two original DNA strands SEPARATE at the nucleotide bases to form two strands that serve as templates.
RNA primer binds to template
DNA polymerase ADDS complementary DNA nucleotides( An adenine (A)on one strand always pairs with a thymine (T) on the opposite strand, and a guanine (G)on one strand always pairs with a cytosine (C) on the opposite strand.
DNA ligase seals the gaps between the Okazaki fragments
TWO identical DNA daughter molecules, each consisting of one old (original) strand and one newly-synthesized strand.
One factor that stops DNA from being copies incorrectly is the nucleotides always match in the same way. Also, an enzyme DNA polymerase checks the arrangement of bases in the new DNA strand, decreasing the chance that DNA copy contains an error.
BIO101 - From One Cell To Two: Cell Division and DNA Replication
This post was originally written in 2006 and re-posted a few times, including in 2010.
As you may know, I have been teaching BIO101 (and also the BIO102 Lab) to non-traditional students in an adult education program for about twelve years now. Every now and then I muse about it publicly on the blog (see this, this, this, this, this, this and this for a few short posts about various aspects of it - from the use of videos, to the use of a classroom blog, to the importance of Open Access so students can read primary literature). The quality of students in this program has steadily risen over the years, but I am still highly constrained with time: I have eight 4-hour meetings with the students over eight weeks. In this period I have to teach them all of biology they need for their non-science majors, plus leave enough time for each student to give a presentation (on the science of their favourite plant and animal) and for two exams. Thus I have to strip the lectures to the bare bones, and hope that those bare bones are what non-science majors really need to know: concepts rather than factoids, relationship with the rest of their lives rather than relationship with the other sciences. Thus I follow my lectures with videos and classroom discussions, and their homework consists of finding cool biology videos or articles and posting the links on the classroom blog for all to see. A couple of times I used malaria as a thread that connected all the topics - from cell biology to ecology to physiology to evolution. I think that worked well but it is hard to do. They also write a final paper on some aspect of physiology.
Another new development is that the administration has realized that most of the faculty have been with the school for many years. We are experienced, and apparently we know what we are doing. Thus they recently gave us much more freedom to design our own syllabus instead of following a pre-defined one, as long as the ultimate goals of the class remain the same. I am not exactly sure when am I teaching the BIO101 lectures again (late Fall, Spring?) but I want to start rethinking my class early. I am also worried that, since I am not actively doing research in the lab and thus not following the literature as closely, that some of the things I teach are now out-dated. Not that anyone can possibly keep up with all the advances in all the areas of Biology which is so huge, but at least big updates that affect teaching of introductory courses are stuff I need to know.
I need to catch up and upgrade my lecture notes. And what better way than crowdsource! So, over the new few weeks, I will re-post my old lecture notes (note that they are just intros - discussions and videos etc. follow them in the classroom) and will ask you to fact-check me. If I got something wrong or something is out of date, let me know (but don't push just your own preferred hypothesis if a question is not yet settled - give me the entire controversy explanation instead). If something is glaringly missing, let me know. If something can be said in a nicer language - edit my sentences. If you are aware of cool images, articles, blog-posts, videos, podcasts, visualizations, animations, games, etc. that can be used to explain these basic concepts, let me know. And at the end, once we do this with all the lectures, let's discuss the overall syllabus - is there a better way to organize all this material for such a fast-paced class.
Today, we continue with the cell biology portion of the course - covering the way cells communicate with each other, something that will come up over and over again for the rest of the course. See the previous lectures:
Continuing with the Thursday BIO101 lecture notes, here is the fifth part. As always, I ask you to correct my errors and make suggestions to make the lecture better. Keep in mind that this is a VERY basic speed-course and that each of the lecture-notes covers roughly 45 minutes (often having 3-4 of these within the same day). This part was first posted on May 14, 2006.
Cell Division and DNA Replication
In the first lecture, we covered the way science works and especially how the scientific method applies to biology. Then, we looked at the structure of the cell, building a map of the cell - knowing what processes happen where in the cell, e.g., the production of energy-rich ATP molecules in the mitochondria.
In the third part of the lecture, we took a closer look at the way DNA code gets transcribed into RNA in the nucleus, and the RNA code translated into protein structure in the rough endoplasmatic reticulum. Finally, we looked at several different ways that cells communicate with each other and with the environment, thus modifying cell function.
All of that information will be important in this lecture, as we cover the ways cells divide, how cell-division, starting with a fertilized cell, builds an embryo, how genetic code (genotype) influences the observable and measurable traits (phenotype) and, finally, how do these processes affect the genetic composition of the populations of organisms of the same species - the process of evolution.
The only way to build a cell is by dividing an existing cell into two. As the genome (the complete sequence of the DNA) is an essential part of a cell, it is necessary for the DNA to be duplicated prior to cell division.
In Eukaryotic cells, chromosomes are structures composed mostly of DNA and protein. DNA is a long double-stranded chain-like molecule. Some portions of the DNA are permanently coiled and covered with protective proteins to prevent DNA expression (transcription). Other parts can be unraveled so transcription can occur.
The number of chromosomes is different in different species. Human cells possess 23 pairs of chromosomes. Prior to cell division each chromosome replicates producing two identical sister chromosomes - each eventually landing in one of the daughter cells.
The process of DNA replication - the way all of the DNA code of the mother cell duplicates and one copy goes into each daughter cell - is the most important aspect of cell division. It is wonderfully described in your handout and depicted in the animation. Other cell organelles also divide and split into two daughter cells. Once the process of DNA replication is over, the new portion of the cell membrane gets built transecting the cell and dividing all the genetic material into two cellular compartments, leading the cell to split into two cells.
Meiosis is a special case of cell division. While mitosis results in division of all types of cells in the body, meiosis results in the formation of sex cells - the gametes : eggs and sperm. Mitosis is a one-step process: one cell divides into two. Meiosis is a two-step process: one cell divides into two, then each daughter immediately divides again into two, resulting in four grand-daughter cells.
Each cell in the body has two copies of the entire DNA - one copy received from the mother, the other from the father. Fertilization (fusion of an egg and a sperm) would double the chromosome number in each generation if the egg and sperm cells had the duplicate copy. Meiosis ensures that gametes have only one copy of the genome - a mix of maternal and paternal sequences. Such a cell is called a haploid cell.
Once the egg and a sperm fuse, the resulting zygote (fertilized egg) again contains double dose of the DNA and is called a diploid cell. Thus the resultant zygote inherits genetic material from both its father and its mother. All the cells in the body except for the gametes are diploid. Sexual reproduction produces offspring that are genetically different from either parent.
DNA replication is a complex process of duplication of the DNA involving many enzymes. It is the first and the most important process in cell division. Please read the handout (BREAKFAST OF CHAMPIONS DOES REPLICATION by David Ng) to appreciate the complexity of the process, but you do not need to memorize any of the enzymes for the exams. Also, it will help your understanding of the process if you watch this animation.
Structures that are created by unwinding of the double helix at a replication origin, from which DNA synthesis will progress in opposite directions.
Short DNA sequences (at least 200 bp) with a high occurence of CpG dinucleotides (for example, a ratio of CpG observed to CpG expected >0.6). In vertebrates, these sequences are often present near the transcription initiation sites of housekeeping genes.
(G4s). Guanine-rich sequences that form a four-stranded DNA structure with tetrads of guanine stacking on top of each other. These have been well studied in telomere regions.
A loose or partly decondensed chromatin structure, found in euchromatin regions that are permissive for transcription.
Proliferating cell nuclear antigen
(PCNA). A homotrimer that is involved in the processivity of DNA polymerases during DNA replication, as well as having a role in DNA repair.
A method in which single DNA molecules are stretched on silanized glass. This method allows the detection of genomic abnormalities such as DNA rearrangements and is also a powerful technique for detecting the spacing between replication origins and the replication fork speed.
A protein complex that mediates the cohesion between the sister chromatids resulting from DNA replication and is also involved in their segregation during mitosis.
A method to detect interactions between chromatin domains in the nucleus.
Extrachromosomal DNA molecules that are able to replicate autonomously in the cell and persist without being integrated in the chromosomes. They can permit retention and expression of transgenes.
Doing the Key Experiment
Frank and Matt
In the fall of 1954, we were reunited in Cal Tech and lived for about eight months in the same house across the street from the lab. We finally could begin doing experiments to test models of DNA replication. It should be noted that DNA replication was our "side" project we also had our "main" projects under the supervision of our respective professors. However, faculty at Cal Tech was kind in allowing two young scientists to venture forward with their own ideas.
While the general experimental approach that took form under the "gin and tonic tree" was straightforward, choices had to be made in how exactly to do the experiment. What organism should we use? Would a chemical trick of making DNA heavier or lighter work and could we measure a small density difference between the two? It took us a while to get the conditions right, about two years.
We first decided to examine the replication of the bacteriophage T4 inside of the bacterium Escherichia coli. Bacteriophages are viruses that invade and replicate inside of a bacterium when new viruses are made, they will burst the bacterium and then spread to new hosts. Bacteriophage have small genomes and are therefore the smallest replicating systems. Frank's PhD thesis was on T4, so he knew how to work with this phage. Max Delbrück and others at Cal Tech were also actively studying phage (see the Narrative on Mutations by Koshland ). Thus, T4 seemed the obvious choice. To create DNA of heavier density than normal DNA, we decided to use the analogue, 5-bromouracil, of the base thymine, in which a heavier bromine atom replaces a lighter hydrogen atom. During replication, 5-bromouracil could be incorporated into DNA, instead of thymine.
However, while this approach seemed reasonable, it did not work in practice. Although we did not appreciate it at the time, during phage growth, the DNA molecules undergo recombination, joining parental DNA to newly synthesized DNA in a manner that after several generations would give no clear-cut distribution of old DNA among progeny molecules. Also, we learned from a recent paper that 5-bromouracil was mutagenic and made a detour into studying mutagenesis before coming back to our main project.
We clearly needed a new strategy.
Instead of phage, we decided to study the replication of the bacterial genome. This was a good choice – the bacterial DNA gave a very sharp and clear band when centrifuged in a solution of cesium chloride (to learn more about why we used cesium chloride to create a density gradient and the general use of this technique see Dig Deeper 3 ).
We also switched our density label. DNA is made up of several elements – carbon, nitrogen, oxygen, phosphorus, and hydrogen. Some of these elements come in different stable isotopes, with atomic variations of molecular weight based upon different numbers of neutrons. Nitrogen-15 (15N) is a heavier isotope of nitrogen (the most common isotope, 14N, has a molecular weight of 14 Daltons). We could easily buy 15N in the form of ammonium chloride (15NH4Cl), which was the only source of nitrogen in our growth medium. The 15N in the medium then found its way into the bacterial DNA (as well as other molecules) in a harmless manner and did not impair bacterial growth.
We also had good luck in that Caltech bought a brand new type of ultracentrifuge called an analytical ultracentrifuge (Model E) developed by the Beckman Instrument Company. The Model E was a massive machine about the size of a small delivery truck (the current model is just a bit bigger than a dishwasher). Importantly, the Model E could shine a UV light beam on the tube while the centrifuge was spinning and detect and photograph the position of the DNA. The good news was that 15N-containing DNA and 14N-containing DNA could be clearly distinguished by their different density positions ( Figure 8 ).Figure 8 Centrifugation of DNA produced either with a normal nitrogen source (14NH4Cl) or a heavy nitrogen food source (15NH4Cl). The DNA molecules (14N-DNA or 15N-DNA) made by the bacteria under these conditions form bands at distinctive densities of cesium chloride during centrifugation. The DNA position was detected with a camera that photographed the sample as it was spinning.
Finally, we had everything in place to try our experiment properly. I decided to set up our first experiment in the following two ways:
1) Grow the bacteria in "light" nitrogen medium and then switch to "heavy."
2) Grow another culture of bacteria in "heavy" nitrogen for many generations and then switch to "light."
Frank was called to a job interview and could not perform this first experiment with me. But before leaving, he warned me – "Don't do the experiment in such a complicated way on your first try. You might mix up the tubes."
I ignored Frank's advice and did the experiment both ways.
In the first experiment after transferring bacteria grown in heavy nitrogen (15N) growth medium and then switched to "light" (14N) nitrogen medium, I saw three discrete bands corresponding to old, hybrid, and new DNA, as predicted by Semi-Conservative replication. Excited developing the photograph in the darkroom, I remember letting out a yelp that caused a young woman working nearby to leave in a hurry. But later I realized my mistake. Frank had been prophetic. I indeed had mixed tubes, combining two different samples, one taken before and the other taken after the first generation of bacterial growth in the light medium. As described for the correct experiment below, there is no time when old, hybrid, and fully new DNA are present at the same time.
When I came back from my trip, Matt and I performed what proved to be the decisive experiment. We grew bacteria in "heavy" nitrogen (15N from 15NH4Cl) and then switched to "light" nitrogen (14N 14NH4Cl) and, at different time points, collected the bacteria by centrifugation, added detergent to release the DNA, and combined this with concentrated CsCl solution to reach the desired density. After 20 hours of centrifugation and the final density positions of the DNAs had come into view, we knew that we had a clean answer ( Figure 9 ). The DNA from bacteria grown in heavy nitrogen formed a single band in the gradient. However, when the bacteria were shifted to a light nitrogen medium and then allowed to replicate their DNA and divide once (first generation), essentially all DNA had shifted to a new, "intermediate" density position in the gradient ( Figure 9 ). This intermediate position was half-way between the all heavy and all light DNA. At longer times of incubation in light nitrogen, after the cells had divided a second time (second generation), a DNA band at lighter density was seen and there were equal amounts of the intermediate and light DNA.Figure 9 Results from the Meselson–Stahl experiment. The positions of DNA bands are shown at four time points. Bacteria were grown in "heavy" nitrogen (15N) and then switched to "light" nitrogen (14N). 0 Generation reflects the time immediately after the nitrogen switch 1.1 and 1.9 generations correspond approximately to the first and second doubling of the E. coli population.
Explorer's Question: Which of the three models (Conservative, Semi-Conservative, or Dispersive) is most consistent with the results of this experiment?
Answer: The Semi-Conservative model. The Conservative model predicts a heavy and light band at the first generation, not an intermediate band. The Dispersive model predicts a single intermediate band at both the first and second generations (the band shifting toward lighter densities with more generation times).
Explorer's Question: Why are the two DNA bands at the 1.9 generation time point of approximately equal intensity?
Answer: After the first generation, each of the two heavy strands is partnered with a light strand. The bacterial DNA consists of one heavy strand and one light strand. When that heavy–light DNA replicates again in the light medium, the heavy strands are partnered with new light strands (intermediate density DNA) and the light strands are also partners with new light strands (creating all light density DNA).
The experiment that Frank described above took hardly any time at all (2 days) and yielded a clean result. We then repeated it without any problem. Once we knew how to set up the experiment, it was relatively easy. But it took us two years of trials before we got the experimental design and conditions right for the final ideal experiment.
The experiment clearly supported the Semi-Conservative Replication model for replication and, in doing so, also supported the double helical model of DNA itself. However, we wanted to do one more experiment that would examine whether the "intermediate" density band of DNA in the first generation was truly made of two and just two distinct subunits, as predicted by the Watson–Crick model. The model predicts that one complete strand of DNA is from the parent and should be heavy and the other complete DNA strand should be all newly replicated and therefore light ( Figure 10 ). We could test this hypothesis by separating the subunits with heat and then analyzing the density and molecular weight of the separated subunits by equilibrium density-gradient ultracentrifugation.Figure 10 Predictions for the Semi-Conservative model for the composition of individual DNA strands after one round of replication.
On the other hand, the Dispersive Model predicted that each DNA strand of first generation is an equal mixture of original and newly replicated DNAs ( Figure 11 ).Figure 11 Predictions for the Dispersive model for the composition of individual DNA strands after one round of replication.
The results from the experiment were again clear ( Figure 12 ). The "intermediate density" DNA in the first generation split apart into a light and heavy component. From the width of the DNA band in the gradient (see Dig Deeper 3 ), we could also tell that the light and heavy DNA obtained after heating had each half of the molecular weight of the intermediate density DNA before heating. These results indicated that each parental strand remained intact during replication and produced a complete replica copy. This was decisive evidence against the Delbrück model for it predicted that both strands would be mosaics of heavy and light, not purely heavy and purely light. And the finding that the separated heavy and light subunits each had half the molecular weight of the intact molecule indicated that DNA was made up of two chains, as predicted by the Watson Crick model, and was not some multichain entity.
Based upon the results in Figure 9 and Figure 12 , we concluded that:
1) The nitrogen of a DNA molecule is divided equally between two subunits. The subunits remain intact through many generations.
2) Following replication, each daughter molecule receives one parental subunit and one newly synthesized subunit.
3) The replicative act results in a molecular doubling.
These conclusions precisely aligned with the Watson–Crick Semi-Conservative model for DNA replication. DNA, as a double-stranded helix, unwinds, and each strand serves as a template for the synthesis of a new strand.
DNA replication . The double helix is unwound and each strand acts as a template for the next strand. Bases are matched to synthesize the new partner strands. DNA replication is the process of producing two identical replicas from one original DNA molecule.
The cell cycle or cell-division cycle is the series of events that take place in a cell leading to its division and duplication (replication) that produces two daughter cells.
- Friday, November 22
- Reading - Chapter 13 (pages: 257-282)
- Chi Square (Bozeman)
- DNA Structure and Replication
- DNA Replication: The Cell's Extreme Team Sport
- The Structure of DNA
- Bozeman - DNA Replication
- DNA Replication
- What happens when your DNA is damaged
- POGIL: DNA structure and replication
- Gave Students a chance to catch up on work
- Reading: Chapter 9 (pages: 186-201) - Worksheet
- The cell cycle (Cell Cycle Phases)
- Mitosis: Splitting Up is Complicated - Crash Course Biology #12
- Bozemena Mitosis
- DNA Warm up Quizizz
- Reading - Chapter 10 (Pages: 204-216)
- Essay Quiz
- Reading Quiz on DNA
- Amoeba Sisters: Meiosis
- Bozeman Mitosis Vs Meiosis
- Reading Quiz on Mitosis and Meiosis
- Grade Essay Quizzes
- DNA Projects are due
- TEST! (Study Guide )
- . he goes over the cell cycle and check points in a very simple and concise manner. . I could watch these vids just to see them drawing - I find it fascinating! The info is good but nothing really on CDK's . a little low key but he goes over the information very well
Alternation of generations: Please review the below links. The important information for this test is knowing at what stages mitosis and meiosis are happening and what they are creating (sporophytes, megaspores, microspores, or gametes)
Look up what organisms produce asexually and sexually. Know the basic way they do it. For example, some plants reproduce asexually from "runners" while they can still reproduce sexually throgh the alternation of generations. Animals are most commonly known to reproduce sexually however some reproduce through budding or parthenogenesis. I don't expect you to know the major details just the very basics.
Review Lecture - Replication of DNA in the chromosomes of eukaryotes
The evidence that each chromatid of a eukaryotic organism contains only one DNA double helix comes from a variety of observations. It begins with the autoradiographic demonstration by J. H. Taylor that tritiated thymidine, incorporated into chromosomes during one round of DNA synthesis, is present in both chromatids at the first division after labelling, but in only one chromatid after a further round of DNA synthesis accomplished in the absence of label. Further evidence comes from those experiments which demonstrate that when two sister chromatids break and fuse one with the other, each chromatid behaves as though it contained two chains of opposite polarity, fusion between chains being restricted to those of like polarity. J. G. Gall’s study of the kinetics of digestion of lampbrush chromosomes by pancreatic DNase also supports the view that each chromatid contains only two polynucleotide chains which are cleaved by this enzyme independently of one another while O. L. Miller’s observations on the dimensions of the fibres remaining after lampbrush chromosomes have been digested by trypsin only allow for there being two polynucleotide chains per chromatid. By means of the technique of DNA fibre autoradiography devised by J. A. Huberman and A. D. Riggs, the units involved in replicating the chromosomal DNA of somatic cells of Xenopus have been compared with those of Triturus. Both these organisms have initiation points for DNA replication that are arranged in tandem, and from each initiation point replication proceeds in opposite directions at divergent forks. The intervals between initiation points in Xenopus range from about 20 to 125 µm apart, whereas those of Triturus are much more widely separated. At 25 °C replication of DNA in Xenopus somatic cells proceeds at 9 µm per hour one-way at each fork, whereas the corresponding rate in Triturus is 20 µm per hour. Triturus somatic cells take about 4 times longer than comparable cells of Xenopus to replicate their DNA. The Triturus genome contains about 10 times as much DNA as the Xenopus genome, and comparison of the replication process in these two organisms indirectly adds weight to the view that the Triturus genome is 10 times longer than that of Xenopus, rather than that it contains 10 times as many DNA double helices per chromatid. DNA fibre autoradiography has also been used to study replication in Triturus spermato-cytes. The round of DNA synthesis just before meiosis in Triturus is an exceptionally long-drawn-out process, taking 9 to 10 days for completion at 16 °C. This lengthy S-phase is not occasioned by abnormally slow replication, the rate being 12 µm per hour one-way at 18 °C, nor is it the result of an exceptional staggering of replication starts. Instead it appears to be correlated with a gross reduction in the number of initiation points for replication. i.e. with an increase in the lengths of the replicating units. A rough calculation suggests that each meiotic chromomere may correspond to a unit of replication during the pre-meiotic S-phase.
Watch the video: Biology 1A Lecture 16 DNA replication, PCR, forensics Free Download u0026 Streaming Internet Arc (January 2022).